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Bioscience Horizons Advance Access published online on April 23, 2008
Bioscience Horizons, doi:10.1093/biohorizons/hzn013
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© Oxford University Press 2008

Sphingosine-1-phosphate activation of TRPC5 in vascular smooth muscle cells

Samantha Fahy

University of Leeds, Leeds, UK

Supervisor: Prof. David Beech, Institute of Membrane and Systems Biology, University of Leeds, Leeds, UK.


   Abstract
Top
 Abstract
Introduction
 Methods and materials
 Results
 Discussion
 Funding
 References
 
Calcium signalling is a complex and diverse system utilized in many cellular processes and in the transmission of cellular information. A number of transient receptor potential (TRP) proteins have been identified in humans and other mammals; these proteins are implicated as having a role in calcium signalling. TRPC5 is a member of this protein family which combines with TRPC1 to form non-selective cation channels in human saphenous vein cells, a type of smooth muscle cell. The exact function of TRPC5 remains elusive, however, it can be activated by sphingosine-1-phosphate (S1P), an endogenous signalling phospholipid involved in SMC migration.

The aim of these experiments was to investigate the effects of S1P on the intracellular calcium concentration, [Ca2+]i, in HSV cells, utilizing dominant-negative (DN)-TRPC5 transfected cells to establish the role played by TRPC5 in this response. A secondary aim was to establish the effect of SMC migration on the above response parameters to S1P using the HSV scratch assay, where a ~2 mm line of cells was scraped away from the surface of a glass cover slip and the remaining cells incubated for 24 h.

Concurrent with the literature, S1P evoked a significant response in HSV cells (n=23; P=0.001). The baseline was significantly lower in the DN-TRPC5 cells compared with the control cells (P<0.001), and the maximum response in the DN-TRPC5 transfected HSV cells reached only 60% of the maximum response in control cells. This suggested that TRPC5 was involved with maintaining basal [Ca2+]i levels and indicated the proportion of the response for which TRPC5 was responsible.

The response to S1P was significantly larger in migrated (n=7) compared with static (n=11) HSV cells (P=<0.001) and this response was delayed by ~2.3 min; the baseline was also higher in the latter group. This suggested a functional change in the cell following migration that may have been attributable to TRPC5, for example, channel up-regulation.

In conclusion, TRPC5-like channels are responsible for a proportion of the S1P response and are implicated in SMC migration. This highlights potential pharmacological targets for the treatment of atherosclerosis, neointimal hyperplasia and coronary heart disease.

Key words: TRPC5, S1P, saphenous, Fura-2, calcium


   Introduction
Top
 Abstract
 Introduction
Methods and materials
 Results
 Discussion
 Funding
 References
 
Calcium signalling is a complex and diverse system, part of many cellular processes and the transmission of cellular information. The main channels permeable to these ions in mammalian cells include stretch-activated channels (SACs) and voltage-, receptor- and store- operated channels (VOCs, ROCs and SOCs, respectively). However, relatively little is known about the mechanisms that underlie and unify this process. A number of transient receptor potential (TRP) proteins have been identified in humans and other mammals;1 these proteins are implicated as having a role in calcium signalling.

TRP proteins are divided into six families; it is the fifth member of the canonical TRP proteins (TRPC5) that will be the main focus here. Sossey-Alaoui et al.2 cloned and characterized human TRPC5 from the encoding gene located on the X chromosome (Xq23). They identified the protein as TRPC5; a novel member of the TRP protein family.

TRPC5 proteins assemble as homomeric or heteromeric tetratamers around a central pore, permeable to cations. TRPC5-like channels can be activated in a number of ways including via receptors.1 For example, a G-protein coupled receptor, such as the endogenous muscarinic M2/3 acetylcholine receptor, is activated by an agonist causing dissociation of the Gβ{gamma} subunit from the G{alpha} subunit. This subunit activates phospholipase (PL) Cβ which causes the hydrolysis of the membrane-bound phosphatidylinositol bisphosphate (PIP2) into the lipophylic diacylglycerol (DAG) and the hydrophilic inositol triphosphate (IP3). The first of these components remains within the membrane, where it is involved in the activation and translocation of a number of proteins including protein kinase (PK)C. The second component travels through the cytoplasm to IP3 receptors (IP3Rs) located on intracellular Ca2+ stores; the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) in muscle cells. Activation of these receptors leads to release of these Ca2+ stores which putatively leads to the opening of SOCs. Zeng et al.3 described TRPC5 channels can be activated by a multiplicity of signals in human embryonic kidney (HEK)293 cells; TRPC5 may be activated as an ROC, as an SOC, by external ionic activation and by intracellular Ca2+.

The exact function of TRPC5 is yet to be defined; evidence suggests a number of possibilities. For example, the location of the gene encoding this protein is associated with certain mental retardation disorders which may suggest a further role.2 This group also suggested its importance in the basic developmental process and polymorphisms of the encoding gene might hinder this development, leading to the aforementioned disorders. Alternatively, Greka et al.4 conjectured that TRPC5 homomer channels have a role in hippocampal growth cone morphology and motility.

This involvement in cell motility has also been suggested for TRPC5 heteromer (TRPC5/TRPC1) channels located in vascular SMCs.5 Sphingosine-1-phosphate (S1P), an endogenous signalling phospholipid, has been demonstrated to have a role in the migration of vascular SMCs.5 Xu et al.5 identified S1P as a novel activator of heteromeric TRPC1/TRPC5 and homomeric TRPC5 channels. S1P concentrations 1, 3 and 10 µM evoked graded responses in HEK293 cells within a 5-min activation period; the median 3 µM was selected for further experiments. As has been previously mentioned, this group conjectured a mechanism for vascular SMC motility, controlled by TRPC5 that was evoked by S1P.

Xu et al.5 used human saphenous vein (HSV) cells, a type of SMC obtained from the saphenous vein in the leg in preparation for coronary artery bi-pass surgery. These cells can be cultured to produce a high-throughput and cost-effective in vitro simulation of physiological conditions. The so-called ‘scratch assay’ was used by this group to study SMC migration; where a line of cells is scraped away from the plated monolayer of cultured HSV cells. Over a 24-h period, the surrounding cells migrated into this scratch; these cells were then identified using fluorescence videomicroscopy and their responses to S1P compared with static cells from the same monolayer. It follows that TRPC5 may be key to the understanding and treatment of a number of vascular diseases including those putatively linked to TRPC1 channel upregulation: atherosclerosis, neointimal hyperplasia and coronary heart disease.6

While S1P application may cause cellular responses such as changes in calcium concentration and cell migration, these responses may not solely be caused by activation of TRPC5-like channels. Xu et al.5 utilized a dominant-negative (DN) transfection to impair the function of TRPC5. This allowed for a comparison to be made between the wild-type (or mock-transfected) proteins and the DN-transfected proteins; where the apparent loss of cellular response was representative of the contribution to the cellular effect made by TRPC5-like channels. The TRPC5-DN transfect is a triple alanine mutation of the conserved sequence of residues leucine, phenylalanine and tryptophan (LFW) located in the channel pore which results in impaired channel function.5

The literature proposes a number of different methods that are used in investigating TRPC5's involvement in Ca2+ signalling. Changes in intracellular Ca2+ concentration, [Ca2+]i, can be measured using conventional fluorescence videomicroscopy and high-throughput, automated fluorometric imaging. Conventional imaging, as it will be referred to here, utilizes videomicroscopy and Ca2+ sensitive dyes to measure and visualize these changes. A xenon light source in a monochromator is used to select the appropriate wavelengths for the fluorescent dye used. Temporally controlled ratiometric imaging exploits the intrinsic properties of duel wavelength ratiometric dyes.

Fura-2 acetoxymethyl ester (AM) is a dual-wavelength ratiometric Ca2+ indicator excited by UV light.7 Unlike Fura-2 sodium and potassium salts, the AM can passively diffuse across cell membranes. The esters are then cleaved by intracellular esterases and the membrane-impermeant Fura-2 remains in the interior of the cell. Fura-2 is a divalent metal ion chelator that has a high affinity for and binds reversibly to Ca2+ at physiologically relevant concentrations; ~100 nm.8 To a lesser extent, Fura-2 may also bind to other bivalent cations such as Mg2+ leading to quenching of the fluorescent signal. Complexation of Fura-2 with Ca2+ causes a conformational change in Fura-2 that is detected through the ratio of the fluorescence intensities at the excitation wavelengths 340 and 380 nm; this indicates the [Ca2+]i. These experiments are carried out under dark conditions so as to prevent bleaching of the fluorescent dye. Frequent illumination of the dye may also lead to bleaching; furthermore, bleaching may give false negative results as it may appear that the [Ca2+]i is decreasing when it is actually the fluorescent signal that is decreasing.

There are several advantages of using such ratiometric dyes, such as Fura-2, over single-excitation dyes, such as Flou 3.8 For example, the intensity of the fluorescence of single excitation dyes is dependent upon the [Ca2+]i while the ratio of the spectral shift produced by ratiometric dyes is proportional to [Ca2+]. Therefore, the exact concentration of the dye does not need to be known as it will not affect the experimental outcome.

The principle aim of my experiments was to investigate the effects of S1P on TRPC5 and TRPC5-like channels in HSV cells. The use of DN-TRPC5 transfected SMC cells would be used identify the components of the S1P response for which TRPC5 is responsible in the cells. A secondary aim of this experiment was to establish the effect, if any, of SMC migration on the above response parameters to S1P.


   Methods and materials
Top
 Abstract
 Introduction
 Methods and materials
Results
 Discussion
 Funding
 References
 
Cell culture
As approved by the Leeds teaching hospital research ethics committee, HSV tissue samples were collected anonymously from patients at Leeds General Infirmary. These patients were undergoing open heart surgery and had given their informed consent. Tissue samples were transported to the laboratory in Hanks' solution (see ‘Materials’) and the median layer of the tissue was extracted by dissection on the day of removal from the host. These cells were then cultured in 75 cm3 flasks in Dulbecco's modified Eagle's medium (DMEM)-F12 media (Invitrogen, UK) supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin (Sigma, UK), and maintained at 37°C in a 5% CO2 incubator. Cells were passaged 1:2 every 3–4 days.

Where indicated, cells were transfected with DN-TRPC5 plasmid using the Basic Nucleofector Kit for primary smooth muscle cells and Amaxa Transfection System (Amaxa Biosystems, Germany). Mock-transfected HSV cells were used as a positive control. The transfection was validated previously and found to be 80–90% effective using the green fluorescent protein.

Passage of HSV cells was carried out by washing with ~2 ml DPBS (Invitrogen, UK) prior to exposure to ~2 ml 1% Trypsin/EDTA (Invitrogen, UK) for 3–7 mins depending upon the intrinsic properties of the cells.

Conventional fluorescence videomicroscopy
At a confluence of ~50%, HSV cells were split onto 10 mm glass cover-slips in 24-well plates and sub-cultured for 24 h prior to imaging. For cells in the scratch assay, a linear scrape of ~2 mm was made with a pipette tip through the monolayer of cells on the cover-slip before being incubated for a further 24 h prior to imaging. Following transfection, cells in the DN paradigm were seeded onto cover-slips 48 h prior to imaging.

Prepared cells were loaded with 3 µM Fura-2 AM (Sigma, UK) in standard bath solution (SBS; see below) for 1 h at 37°C then washed with SBS for a minimum of 30 min at room temperature. Loaded cells were viewed using an inverted microscope (Axiovert 25 CF I; Zeiss, Germany) in combination with a monochromator (Till Photonics, Germany), which selected the excitation wavelength provided by a xenon arc lamp. Cells were continuously perfused with SBS at a rate of ~2 ml/min; following the establishment of a stable baseline with SBS, the perfusion was changed to 3 µM S1P (Sigma, UK), applied at the same rate for the below specified period of time. Cells in the scratch assay were identified as migrated or static depending on their spatial positioning to the visible scratch area. Emissions were collected by a 510 nm filter and an image was taken at each of the excitation wavelengths via a CCD camera (Orca ER; Hamamatsu, Japan) at 10-s intervals. Imaging was controlled using Openlab 4 software (Image Processing & Vision Company Ltd, UK).

High-throughput fluorometric imaging
At a confluency of ~60%, cells were seeded into clear 96-well plates. Following a 24-h incubation period, cells were loaded with 2 µM Fura-2 AM in SBS for 1 h at 37°C before being manually washed three times with SBS. SBS was replaced in each well in varying volumes according to the protocol being followed. Changes in [Ca2+]i in response to application of various substances were read in a 96-well plate FlexStation II (Molecular Devices, USA). Fura-2 was excited at 340/380 nm every 10 s for a total duration of 300 s.

Materials
Hanks' solution contained (in mmol/L): 137 NaCl, 5.4 KCl, 0.01 CaCl2, 0.34 NaH2PO4, 0.44 K2HPO4, 8 D-glucose, 5 HEPES. SBS contained (in mmol/L): 130 NaCl, 5 KCl, 8 D-glucose, 10 HEPES, 1.2 MgCl2 and 1.5 CaCl2, pH titrated to 7.4 and osmolarity corrected to 290 mOsm using D-mannitol where necessary.

Methods of analysis
Recordings from conventional fluorescence videomicroscopy were analysed off-line using Openlab 4. The means of five regions of interest from each cell were taken, as specified below. [Ca2+]i is expressed as the fluorescence intensity ratio of the cells at both excitation wavelengths (340 and 380 nm). For high-throughput fluorometric imaging, real-time data from the Flexstation were collected by SoftMax Pro (Molecular Devices, USA).

All data were analysed using Origin Pro 7.5 (Origin Lab Corporation, USA). Conventional fluorescence microscopy data were expressed as mean ± SEM for n cells (in parentheses); n is number of cells analysed per experiment, parentheses are the number of cover-slips. High-throughput fluorometric imaging data were expressed as mean ± SEM for n cells, where n is the number of wells from which that data set was collated. The statistical significance, if any, was investigated using a one-way ANOVA test for three data sets and two-sample paired or independent Student's t-test for two data sets, P < 0.05 being regarded as significant.


   Results
Top
 Abstract
 Introduction
 Methods and materials
 Results
Discussion
 Funding
 References
 
Figure 1A is an example image of fura-2 AM loaded HSV cells as viewed on the conventional fluorometric imaging rig. Application of S1P to such HSV cells caused a significant change in [Ca2+]i (P < 0.001; Fig. 1B). Following this transient response in the HSV cells, the fluorescence ratio declined towards the baseline.


Figure 1
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  		Figure 1. Establishing a response to S1P in HSV cells using conventional videomicroscopy. (A) Example image showing HSV cells loaded with Fura-2 AM indicator dye. (B) Response to 3 µM S1P in HSV cells n=23(3) where n is the number of cells, parentheses indicate the number of cover slips.

 
As has been previously mentioned, HSV cells contain a number of receptors on which S1P could be acting and the TRPC5-DN transfection allows for information to be gained about the response mediated by TRPC5 and TRPC5-like channels. Figure 2A shows the response to S1P in DN- and mock-transfected HSV cells. The baseline [Ca2+]i was significantly lower in the DN- compared to the mock-transfected cells (P < 0.001). S1P application to the DN-transfected cells caused the transient response, followed by a return to the baseline [Ca2+]i. A transient response was also observed in the mock-transfected cells but the [Ca2+]i at the endpoint was significantly higher than the baseline (P < 0.05). While the amplitude of the responses to S1P was found to be significant (P < 0.001) for both cell types, the maximum response in the DN-transfected cells reached only 60% of the maximum response in the mock-transfected cells; this difference was significant (P < 0.001).


Figure 2
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  		Figure 2. Effect of TRPC5-DN transfection on the response to S1P and methanol in HSV cells. (A) Responses to 3 µM S1P in mock-transfected HSV cells (closed squares ({blacksquare}); n = 24) and in DN-transfected HSV cells (open squares ({square}); n = 24). (B) Responses to methanol in mock-transfected HSV cells (closed squares ({blacksquare}); n = 24) and in DN-transfected HSV cells (open squares ({square}); n = 24).

 
Figure 2B shows the response to methanol in DN- compared to mock-transfected cells. A peak, small in comparison to those for S1P (Fig. 2A), was observed in the mock-transfected cells in response to methanol; this was significant (P = 0.05). A non-significant peak was also observed in the DN-transfected cells following methanol addition.

To allude once more to the ongoing theme; S1P initiates SMC migration and TRPC5 has been implicated as having a role in such migration.5 The purpose of the HSV scratch assay was to establish the differences, if any, in the Ca2+ response to S1P in static cells compared with cells that had migrated into a ~2 mm linear scratch. As a general point of observation, cells located within the scratch were more easily identifiable by a brighter fluorescence compared with other cells (Fig. 3A). This was not considered to be diagnostic and cells were initially deemed to be either ‘static’ or ‘migrated’ by opinion. The initial analysis of results from these two groups resulted in high errors and confusing data (data not shown). Peak responses for each cell were binned according to their values to produce two distinct populations. These populations define the meaning of ‘static’ and ‘migrated’ cells for the purposes of this discussion.


Figure 3
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  		Figure 3. Effect of cell migration on the response to S1P in HSV cells where a ~2 mm line of cells had been scraped from the surface of a glass coverslip and the remainder incubated for 24 h. (A) Annotated example image showing HSV scratch assay with cells loaded with Fura-2 AM indicator dye demonstrating scratch (white perforated line) and putative migrated cells with increased fluorescence compared with other cells (white arrows). (B) Responses to S1P in HSV cells in the scratch assay; cellular responses were binned according to the maximum peak response per cell into these two distinct populations; ‘static’ (closed squares ({blacksquare}); n = 11(4)) and ‘migrated’ (open squares ({square}); n = 7(4)).

 
There were many note-worthy differences between the static and migrated cells, as illustrated in Fig. 3B. The baseline [Ca2+]i was significantly higher in the static compared with the migrated cells (P < 0.001) and the response in the static cells occurred almost immediately after the application of S1P at t = 1.5 min and peaks at t = 2.8 min. There was a change in the [Ca2+]i in the migrated cells around this point which was found to be significant compared with the baseline (P = 0.001), but was small in comparison to the maximum response. The main peak in the migrated cells occurred at t = 5 min; this change in the [Ca2+]i was found to be significant compared with the baseline (P < 0.001). Following the transient response in the static cells, the [Ca2+]i did not return to baseline; this sustained component of the response was significantly higher than the baseline (P < 0.05) and was followed by another small, non-significant peak at t = 7.5 min. Following the transient response in the migrated cells, it was unclear if the [Ca2+]i returned to the baseline. It can, however, be said that the [Ca2+]i in these cells does fall below the sustained phase of the static cells.


   Discussion
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
Funding
 References
 
The above results were sufficient to satisfy the initial aims of the work and, as is fully discussed below, highlighted some intriguing properties of TRPC5-like channels. Consistent with the published data, S1P did elicit a significant response in these cells producing the characteristic transient response that has been previously observed.5 Unlike in the literature,3 it is unclear from Fig. 1B whether or not the S1P response had a sustained component. However, this phase was observed in other experiments on this cell type (data not shown).

While these data show that S1P evoked a response in the form of a change in [Ca2+]i, it could not be unequivocally established from these results if this response was caused by S1P activating TRPC5. The DN-transfection was used to examine the effects of S1P in the absence of functional TRPC5-like channels. The implications of the results from this novel work were 2-fold. First, the lower baseline observed in the DN- compared with the mock-transfected cells suggested that TRPC5-like channels contribute to basal Ca2+ levels. This was in a manner that is independent of activation by agonists such as S1P, suggesting a spontaneous activity of these channels. Secondly, the difference between the amplitudes of the responses to S1P in the DN- compared with the mock-transfected cells is also relevant to this discussion. The maximum response reached in the DN-transfected cells reached only 60% of the maximum response reached in the mock-transfected cells. It seems intuitive that the discrepancy of 40% was due to Ca2+ current mediated by normally functioning TRPC5-like channels. It follows that the response in the DN-transfected cells was mediated by the normally functioning TRPC5-like channels, i.e. those not affected by the transfection and by other endogenous non-selective cation channels activated by S1P. There is a paucity of data in the literature to support this latter conjecture.

The HSV scratch assay is an in vitro simulation of SMC migration that was utilized here to further characterize the response of SMC cells to S1P. A marked difference was observed in the intensity of the fluorescence of the cells located in or near the scratch (Fig. 3A). The relatively small differences in the basal [Ca2+]i (Fig. 3B) are unlikely to account for this higher intensity of fluorescence. It is likely that changes in the cell-shape due to migration affected the fluorescent signal intensity as is seen during blood vessel contraction.9

There are several possible factors that may have given rise to the aforementioned difficulties in identifying whether these cells were migrated or static. It is important to bear in mind that this identification was by opinion and therefore subjective. Following the initial scratch, there was a residual layer of loose cells in the surrounding area. These loose cells were washed away 24 h later when the cells were loaded with Fura-2 AM, however, in the meantime, it is feasible that these multi-layered cells may have acted together in a tissue-like manner. This may have affected the properties of cells contacting the glass-cover slip.

Although this design was the same as that used by Xu et al.,5 this idea highlights a limitation in the experimental design and an area for improvement with this type of work. Currently, there is no equipment available to enable less subjective, controlled measurements of calcium responses in migrated cells. For example, while calcium changes can be automatically measured using an equipment such as the FlexStation, and SMC migration can be measured using Matrigel (BD Biosciences, UK), which is highly valid to the in vivo situation, there is no system that combines the two. Arguably, the human errors experienced with this assay would have been reduced with further practise.

The results from the HSV scratch assay revealed a number of differences between static and migrated SMCs. The higher baseline observed in the static cells suggested a difference in Ca2+ permeabilities between the two cell types in a manner that is independent of channel activation. This finding may suggest down-regulation of a non-selective cation channel in the cell membrane that does not necessarily contain TRPC5. Following on from work by Kumar et al.,6 which suggested that TRPC1 is up-regulated in SMC migration, it is feasible that TRPC5 up-regulation is associated with this phenomenon also. This is backed up by Xu et al.5 who found co-assembly of TRPC1 and TRPC5 sub-units in SMCs and is supported by the results showing that S1P activates TRPC5 (Fig. 2A). This latter point is relevant due to the aforementioned tendency of this phospholipid to induce SMC migration. This could have been explored further by incubating the SMCs in S1P with the aim of causing SMC migration. The effect of this induced migration on the amplitude of the response to S1P could then be compared with responses in cells incubated in SBS.

In agreement with the aforementioned literature, the differences in the amplitudes of the responses between the static and migrated cells may also have been due to differences in channel expression in the migrated cells. Alternatively, migration of SMCs may have increased the affinity of the GPCR for S1P to the effect of causing an increased response. Xu et al.5 suggested that external S1P activation is via activation of a G-protein coupled receptor linked to PLC but that TRPC5 may act as an ionotropic receptor in response to intracellular S1P. Intracellular S1P may have been contributing to this response, causing a difference in the [Ca2+].

The delayed response observed in the migrated cells has not been previously demonstrated and indicates a further functional change following migration. As the amplitude of the response remains the same, it is unlikely that a down-regulation of the GPCR for S1P has occurred but the delay could be due to a decrease in sensitivity to S1P. Indeed these receptors may not be affected at all, but coupling of these receptors to TRCP5-like channels has in some way been altered. A DN-TRPC5 scratch assay may have assisted in finding out the reasons underlying this delay.

Besides the aforementioned technical limitations of the equipment and techniques used for these experiments, it is also important to note that calcium indicator dye experiments are not direct measures of ion-channel function. This is because they only reflect the level of free Ca2+, regardless of the source. Additionally, attention should be drawn to the notion that TRPC5 channels are non-selective cation channels, so the use of Ca2+ indicators may not reflect complete channel function. These experiments serve only as approximations to demonstrate principals based on manipulation of TRPC5 using S1P. To directly test TRPC5 channel function, patch-clamping could have been carried out.

The clinical use of this information about the involvement of TRPC5 is presently minimal. Limited information about the functional role of TRPC5-like channels means that selective targeting of this protein cannot yet be used to pharmacologically prevent SMC migration in vascular disease and other associated conditions. It remains unknown whether there is an association between the X chromosome loci associated with certain mental retardation disorders and the region encoding TRPC5.2 Understandably, a link between these regions of the gene highlights some considerations if TRPC5 is to be targeted pharmacologically. Presently, TRPC5 knowledge can only assist in understanding SMC migration-related disorders.

The experimental data provide strong evidence to suggest that S1P activates TRPC5-like channels in HSV cells. Additionally, that migrated SMC react differently to S1P compared with static cells. This provides a rationale for declaring the aims of the investigation as satisfied and concludes this report.


   Funding
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Funding
References
 
This project was funded by in part by the Faculty of Biological Sciences, University of Leeds and also by the Wellcome Trust.


   Acknowledgements
 
I would like to thank all members of the Beech Lab for their help and support throughout this project, with special thanks to David Beech, Eman Al-Shawaf and Jing Li.


   References
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Funding
 References
 

  1. Clapham DE. TRP channels as cellular sensors. Nature (2003) 426:517–524.[CrossRef][Medline]
  2. Sossey-Alaoui K, Lyon JA, Jones L, et al. Molecular cloning and characterization of TRPC5 (HTRP5), the human homologue of a mouse brain receptor-activated capacitative Ca2+ entry channel. Genomics (1999) 60:330–340.[CrossRef][Web of Science][Medline]
  3. Zeng F, Xu S-Z, Jackson PK, et al. Human TRPC5 channel activated by a multiplicity of signals in a single cell. J Phys (2004) 590:739–750.
  4. Greka A, Navarro B, Oancea E, et al. TRPC5 is a regulator of hippocampal neurite length and growth cone morphology. Nat Neuro (2003) 6:837–845.[CrossRef][Web of Science][Medline]
  5. Xu S-Z, Muraki K, Zeng FA, et al. Sphingosine-1-phosphate-activated Ca2+ channel controlling vascular smooth muscle cell motility. Circ Res (2006) 98:1381–1389.[Abstract/Free Full Text]
  6. Kumar B, Dreja K, Shah SS, et al. Upregulated TRPC1 channel in vascular injury in vivo and its role in Human neointimal hyperplasia. Circ Res (2006) 98:557–563.[Abstract/Free Full Text]
  7. Grynkiewicz G, Peonie M, Tsein RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem (1985) 260:3440–3450.[Abstract/Free Full Text]
  8. Haugland RP. The Handbook 10th Edition. (1995) Carlsbad, CA: Invitrogen Corporation.
  9. Himpens B, Lydrup ML, Hellstrand P, et al. Free cytosolic calcium during spontaneous contractions in smooth muscle of the guinea-pig mesotubarium. Euro J Phys (1990) 417:404–409.[CrossRef]
Submitted on 17 January 2008; accepted on 11 February 2008


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